Determination of Fatty Acids Composition in Polysorbates 80 and 20 Pharmaceutical Raw Materials by HPLC with Mass Detection

Significance of the topic

Polysorbates 80 and 20 are widely used non-ionic surfactants in pharmaceutical, food, and cosmetic formulations for their emulsifying and solubilizing properties. Detailed characterization of their fatty acid profiles is essential for quality control, ensuring batch-to-batch consistency and compliance with pharmacopeial standards. Conventional GC-FID methods require lengthy hydrolysis and derivatization steps. The development of rapid HPLC-MS assays addresses the need for efficient, accurate and direct measurement of free fatty acids in these excipients.

Objectives and study overview

This study aimed to establish and validate novel high-performance liquid chromatography methods with mass spectrometric detection to determine the free fatty acid composition of polysorbate 80 and 20 raw materials. Key goals included:

• Avoidance of methylation and derivatization workflows.
• Direct injection of hydrolyzed samples into HPLC-MS.
• Identification and quantitation of USP-specified fatty acids and positional isomers.

Used methodology and instrumentation

Polysorbate samples were hydrolyzed with 1 M potassium hydroxide at 40 °C for 6 h, neutralized with formic acid and diluted in water/ethanol (50:50, v/v). Following filtration, analytes were separated on a reversed-phase XBridge BEH C18 column (4.6×100 mm, 3.5 μm) at 60 °C using isocratic and gradient programs for PS 80 and PS 20. Mass detection employed negative electrospray ionization with:

• An Arc HPLC system coupled to an ACQUITY QDa detector for SIR quantitation.
• A Xevo G2-XS QTof mass spectrometer interfaced to an ACQUITY UPLC I-Class for accurate mass confirmation.
• Empower Software for data acquisition and USP monograph–compliant calculations.

Main results and discussion

The HPLC-MS methods achieved baseline separation of all USP-specified fatty acids in both polysorbate grades. Single Ion Recording enabled precise quantitation, and QTof accurate mass data facilitated rapid peak identification. Key findings:

• Polysorbate 80: Oleic (18:1) and linoleic (18:2) isomers such as cis-vaccenic, elaidic and conjugated linoleic acids (Δ9,11; Δ10,12) were resolved and quantified.
• Polysorbate 20: Complete separation of caproic to oleic acids in accordance with USP criteria.
• All measured fatty acid percentages in test batches fell within USP-specified limits.

Benefits and practical applications of the method

The direct HPLC-MS approach offers multiple advantages for routine QC laboratories:

• Elimination of derivatization steps reduces sample preparation time.
• Enhanced specificity and sensitivity through mass detection.
• Integration with compliant data systems supports streamlined reporting.
• Capability to detect additional isomeric impurities not covered by GC-FID.

Future trends and potential applications

Ongoing trends are expected to further improve analytical workflows:

• Miniaturized UHPLC-MS platforms for higher throughput.
• Advanced columns with sub-2 μm particles for faster separations.
• Automated sample hydrolysis and on-line dilution modules.
• Expanded application to stability assessments and degradation profiling of excipients.

Conclusion

The described HPLC-MS procedures provide a rapid, accurate and comprehensive solution for fatty acid profiling in polysorbates 80 and 20. By bypassing derivatization and leveraging mass spectrometric specificity, these methods enhance quality control efficiency and enable detection of isomeric species beyond conventional GC-FID capabilities.

References

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3. United States Pharmacopeia (USP). Polysorbate 80 Monograph, USP–NF 2021 Issue 1. The United States Pharmacopeia Convention; Official May 1, 2020.
4. United States Pharmacopeia (USP). Polysorbate 20 Monograph, USP–NF 2021 Issue 1. The United States Pharmacopeia Convention; Official May 1, 2020.
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